The long-term goal of this project is to understand the developmental and coordinate regulation of globin genes in erythroid cells. These studies provide therapeutic strategies for genetically inherited dysfunctions of erythroid cells such as thalassemias and sickle cell anemia. Previous efforts on this project culminated in the detailed comparison of alpha- like and beta-like globin gene clusters between rabbit and human that revealed several candidates for regulatory elements. (1) Despite the requirement to balance production of alpha- and beta-globins, their genes reside in radically different segments of the genome. The CpG islands encompassing the rabbit alpha-globin gene may form an enhancer- independent promoter, and this will be tested by measuring the levels of expression in cells transfected with a series of constructs varying size, number and composition of CpG islands, and by attempting to resurrect an inactive CpG island by addition of active 5' flanking sequences. The ability of fragments of the alpha-globin gene to serve as enhancers will be tested, sequence-specific binding proteins will be purified, and cDNA clones encoding them isolated to aid in structural characterization. These proteins will be compared with those enriched in the CpG-rich fraction of chromatin. (2) Strong regulatory elements located distally in the rabbit beta- and alpha-like globin gene clusters (called locus control regions) will be sequenced, compared to homologous regions in humans and other mammals, and tested for enhancer function, independence from position effects, impact on developmental control and scaffold attachment. (3) Finally, striking patterns of sequence matches are seen in comparisons of rabbit and human epsilon-globin genes and the enhancer located 3' to the beta-globin gene. These conserved sequences will be tested for ability to affect expression of linked reporter genes and to bind nuclear proteins specifically. Novel proteins will be purified and characterized.